SARS-CoV-2 Delta Variant N Gene Mutations Reduce Sensitivity to the TaqPath COVID-19 Multiplex Molecular Diagnostic Assay

Abstract

As the SARS-CoV-2 virus evolves, mutations may result in diminished sensitivity to qRT-PCR diagnostic assays. We investigated four polymorphisms circulating in the SARS-CoV-2 Delta lineage that result in N gene target failure (NGTF) on the TaqPath COVID-19 Combo Kit. These mutations were detected from the SARS-CoV-2 genome sequences that matched with the diagnostic assay results of saliva specimens. Full length N genes from the samples displaying NGTF were cloned into plasmids and assayed using three SARS-CoV-2 qRT-PCR assays. These constructs resulted in reduced sensitivity to the TaqPath COVID-19 Combo Kit compared to the controls (mean Ct differences of 3.06, 7.70, 12.46, and 14.12), but were detected equivalently on the TaqPath COVID-19 Fast PCR Combo 2.0 or CDC 2019_nCoV_N2 assays. This work highlights the importance of genomic sequencing to monitor circulating mutations and provide guidance in improving diagnostic assays.

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“…Since dPCR relies on sequence homology between probes, primers, and template nucleic acids, it is susceptible to failure as SARS-CoV-2 evolves mutations. There have been numerous reports of SARS-CoV-2 mutations that cause failure on diagnostic RT-PCR assays ( 26 – 28 ). Mutations in other nucleotides of the probe or primer binding regions will result in failed amplification, as observed with the K444 reference probe against the XBB lineage ( Fig.…”

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